VCorrections

Correction. In the article "Restoration of Ca2" influx and de-granulation capacity of variant RBL-2H3 cells upon implanta-tion of isolated cromolyn binding protein" by N. Mazurek, P.Bashkin, A. Loyter, and I. Pecht, which appeared in number19, October 1983, of Proc. Natl. Acad. Sc. USA (80, 6014-6018),an error in the printing process resulted in faulty reproductionof portions of the abstract and the introduction. This materialis reproduced-below;

ABSTRACT Recently, variants of the rat basophilic leukemiacells (RBL-2H3), deficient in their binding capacity for. the anti-allergic drug cromolyn-but displaying unimpaired ability to bindIgE, were selected and cloned [Mazurek, N., Bashkin, P., Pe-trank, A. & Pecht, I. (1983) Nature (London) 303, 528-530]. Al-though the histamine content and the number of IgE receptors inthese variants are similar to those of the parental cells, they can-not be stimulated immunologically to allow Ca2+ influx and to de-granulate. In contrast, the Ca2+ ionophore A23187 causes thesevariants to degranulate, indicating that the- mechanism distal tothe Ca2+ gating is intact in- the variants. The cromolyn bindingprotein (CBP), present in the membranes of RBL-2H3 cells, hasrecently been isolated by affinity chromatography under non-denaturing conditions. In the current study we have used Sendai-virus envelopes as fusogenie carriers to implant the purified CBPinto the membrane of variant basophils that were defective in it.This fusion leads to the restoration of Ca2+ uptake and degran-ulation capacity of the variants after IgE-mediated stimulation.These restored activities seem to show a sigmoidal dependence onthe amount of incorporated CBP. Saturation values comparableto those of the parental line are reached when the level of im-planted CBP approaches its density on the latter line. The re-stored capacity is due to the implanted CBP, because the rein-stated immunological response-can be blocked by the inhibitorydrug cromolyn and by monoclonal antibodies specific to CBP, bothshown to prevent Ca.+-uptake and degranulation in mast cells andparental RBL-2H3- cells. These results point out that CBP playsan important role in the Ca2+ gating process resulting in degran-ulation.

Mast cells and a basophilic tumor analog, the rat basophilic leu-kemia cell line (RBL-2H3), have been increasingly used as amodel system for analysis of the cellular mechanism involvedin secretion by exocytosis (1). Like most secretory cells, they.use calcium ions as a mediator in stimulus-secretion coupling(2, 3). In addition, degranulation is of clinical significance; be-cause of its involvement in -the allergic reaction (4,.5). The di-sodium salt of 1,3-bis(2-carboxychromon-5-yloxy)-2-hydroxy-propane, cromolyn, inhibits Ca2+ uptake and hence the releaseof anaphylactic mediators from mast cells and has found wideapplication in the prophylactic treatment of allergic bronchialasthma (6-9). The drug has been shown to chelate alkaline earthions in low polarity medium with a clear preference of Ca2+over Mg2+ (10, 11). Moreover, a specific CaO'-dependent bind-ing site, through which the pharmacological behavior of cromo-lyn is expressed, has been demonstrated on the surface mem-brane of mast cells and RBL-2H3 cells (12). We have recentlyisolated under nondenaturing conditions a cromolyn bindingprotein (CBP) (13). This protein was found to consist of a singlesubunit of 60,000 daltons, to contain 20-25% acidic amino acids,and to display a low pI value of 4-4.5.

Proc. Nati. Acad. Sci. USA 80 (1983) 7373

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Proc. Nati. Acud. Sci. USAVol. 80, pp. 6014-6018, October 1983Immunology

Restoration of Ca2+ influx and degranulation capacity of variantRBL-2H3 cells upon implantation of isolated cromolynbinding protein

(antiasthmatic drug/histamine release/membrane fusion/Sendai virus/basophils)

N. MAZUREK*, P. BASHKIN*, A. LOYTERt, AND I. PECHT**Department of Chemical Immunology, The Weizmann Institute of Science, Rehovot 76100, and tDepartment of Biological Chemistry, Institute of Life Sciences,The Hebrew University of Jerusalem, Jerusalem, Israel

Communicated by Ephraim Katchalski-Katzir, June 21, 1983

ABSTRACT Recently, variants of the rat basophilic leukemiacells (RBL-2H3), deficient in their binding capacity for the anti-allergic drug cromolyn but displaying unimpaired ability to bindIgE, were selected and cloned [Mazurek, N., Bashkin, P., Pe-trank, A. & Pecht, I. (1983) Nature (London) 303, 528-530]. AL-though the histamine content and the number of IgE receptors inthese variants are similar to those of the parental cells, they can-not be stimulated immunologically to allow Ca2" influx and to de-granulate. In contrast, the Ca2' ionophore A23187 causes thesevariants to degranulate, indicating that the mechanism distal tothe Ca2' gating is intact in the variants. The cromolyn bindingprotein (CBP), present in the membranes of RBL-2H3 cells, hasrecently been isolated by affinity chromatography under non-denaturing conditions. In the current study we have used Sendai-virus envelopes as fusogenic carriers to implant the purified CBPinto the membrane of variant basophils that were defective in it.This fusion leads to the restoration of Ca2' uptake and degran-ulation capacity of the variants after IgE-mediated stimulation.These restored activities seem to show a sigmoidal dependence onthe amount of incorporated CBP. Saturation values comparableto those of the parental line are reached when the level of im-planted CBP approaches its density on the latter line. The re-stiored capacity is due to the implanted CBP, because the rein-stacf immunological response can be blocked by the inhibitorydrq5 cromolyn and by monoclonal antibodies specific to CBP, bothshown to prevent Ca2' uptake and degranulation in mast cells andparental RBL-2H3 cells. These results point out that CBP playsan aiportant role in the Ca2+ gating process resulting in degran-UlaVIDn.

Ma5t-cells and a basophilic tumor analog, the rat basophilic leu-kemia cell line (RBL-2H3), have been increasingly used as amodel system for analysis of the cellular mechanism involvedin secretion by exocytosis (1). Like most secretory cells, theyuse calcium ions as a mediator in stimulus-secretion coupling(2, 3). In addition, degranulation is of clinical significance, be-cause of its involvement in the allergic reaction (4, 5). The di-sodium salt of 1,3-bis(2-carboxychromon-5-yloxy)-2-hydroxy-propane, cromolyn, inhibits Ca2+ uptake and hence the releaseof anaphylactic mediators from mast cells and has found wideapplication in the prophylactic treatment of allergic bronchialasthma (6-9). The drug has been shown to chelate alkaline earthions in low polarity medium with a clear preference of Ca2+over Mg2' (10, 11). Moreover, a specific Ca2+-dependent bind-ing site, through which the pharmacological behavior of cromo-lyn is expressed, has been demonstrated on the surface mem-brane of mast cells and RBL-2H3 cells (12). We have recently

isolated under nondenaturing conditions a cromolyn bindingprotein (CBP) (13). This protein was found to consist of a singlesubunit of 60,000 daltons, to contain 20-25% acidic amino acids,and to display a low pI value of 4-4.5.To examine the physiological role of CBP, we have selected

RBL-2H3 cell variants deficient in cromolyn binding but dis-playing unimpaired ability to bind IgE (14). These variants, al-though similar to the parental cells in histamine content, werenot stimulated. to allow Ca2" influx and degranulate in an IgE-mediated reaction. However, the Ca2+ ionophore A23187, knownto circumvent the initial membranal processes, caused thesevariants to degranulate in a noncytotoxic manner (14). Theseobservations indicate that the mechanism distal to the Ca2+ gateis intact in the variants deficient in cromolyn binding.The role played by CBP in the degranulation process, and

particularly in the Ca2' influx, was now directly probed by in-corporating the isolated CBP into the membrane of the cro-molyn-binding-defective variants. This fusion, described in thepresent study, restores the capacity for Ca2" uptake and de-granulation after immunological stimulation of the variant cells,up to the level observed in the parental RBL-2H3 cell line.

MATERIALS AND METHODSBuffers. The following buffers were used: resolution buffer

(0.1 M NaCl/50 mM Tris HCl/0.1 mM phenylmethylsulfonylfluoride, pH 7.4); reconstitution buffer (10 mM Tris-HCl/2 mMCaCl2/2 mM MgSO4/0.02% NaN3/0.1mM phenylmethylsul-fonyl fluoride, pH 7.4); Tyrode solution (137.0 mM NaCl/2.7mM KCl/0.4 mM NaH2PO4/10 mM Hepes/5 mM glucose/1.8mM CaCl2/1.0 mM MgC12). The medium used in the exper-iments was RPMI 1640 (GIBCO).

Antisera. Antibodies against Sendai-virus envelope glyco-proteins were raised in rabbits, as described (15). Monoclonalanti-CBP antibodies were generated by hybridoma cells, ob-tained by fusion of NS1 cells (nonsecreting murine myeloma)with spleen lymphocytes from mice immunized with fixed RBL-2H3 cells, following the procedure of Galfre et al. (16). Su-pernatants of hybridoma cultures were screened for inhibitionof radiolabeled cromolyn binding to RBL-2H3 cells. Five pos-itive secreting cultures were obtained and double cloned onsoft agar. Inhibition of 95% of Ca2+ uptake and degranulationwas observedwhen RBL-2H3 cells were preincubatedwith thosemonoclonal anti-CBP antibodies prior to immunological stim-ulation. Monoclonal antibodies that failed to inhibit radiola-

Abbreviations: CBP, cromolyn binding protein; V vesicles, reconsti-The publication-costs of this article were defrayed in part by page charge tuted Sendai-virus envelopes; VCBP vesicles, CBP integrated into Vpayment. This article must therefore be hereby marked "advertise- vesicles; DNP, 2,4-dinitrophenyl; (DNP)8-BS albumin, a conjugate ofment" in accordance with 18 U.S.C. §1734 solely to indicate this fact. eight molecules of DNP per one molecule of bovine serum albumin.

>kaC~c kUS fAWo (\cV3) 7?373

Proc. Natl. Acad. Sci. USA 80 (1983) 6015

beled cromolyn binding to RBL-2H3 cells had no effect on Ca2"uptake and degranulation and served as controls in the aboveexperiments (unpublished data).Tumor Cells. RBL-2H3 cells were grown in stationary flask

cultures as described (13). Variant subelones (RBL-2H3, E7-14,and E7-23) deficient in CBP and incapable of degranulation weresorted out from the RBL-2H3 parental line by using the flu-orescence-activated cell sorter (FACS II, B. D. Electronics,Sunnyvale, CA) as described elsewhere (14).

Isolation of CBP. CBP was isolated from membranes of RBL-2H3 cells grown as intraperitoneal solid tumors, induced by in-jection of their suspension into 2-wk-old WF/Mai Wistar-Furthrats. The isolation procedure of the protein has been previouslydescribed (13). Purified CBP was stored in Tyrode buffer con-taining 0.2% Triton X-100 at -700C.

Preparation of Vesicles. Two types of vesicles were pre-pared: (i) solubilized Sendai-virus envelope vesicles (V vesicles)and (ii) solubilized Sendai-virus envelopes containing CBP (VCBPvesicles). V vesicles were prepared by solubilization of Sendaivirus (20 mg of protein) in 2 ml of resolution buffer containing2% Triton X-100 for 1 hr at room temperature. The turbid so-lution was then centrifuged (1 hr, 100,000 X g) to yield a clearsupernatant containing mainly the two envelope glycoproteinsNH and F (neuraminidase/hemagglutinin glycoprotein and thefusogenic protein) (17). For preparation of VCBP vesicles, iso-lated CBP (50 Ag of protein per 0.3 ml of resolution buffer con-taining 1% Triton X-100) was mixed with the solubilized viralenvelopes (5 mg of protein per 0.7 ml of resolution buffer) anddialyzed in Spectrapor membrane-2 against a 1,000-fold excessof reconstitution buffer containing 1 g of bovine serum albuminper liter (for efficient removal of the detergent). V vesicles wereprepared by the same procedure, except that CBP was omittedand replaced by resolution buffer alone. Dialysis was continuedfor 96 hr at 4°C until the Triton X-100 concentration droppedbelow 0.02% (17). The dialyzed solutions were centrifuged at100,000 x g for 3 hr at 4°C to obtain reconstituted vesicles. Thepellet was suspended in 0.3-0.5 ml of resolution buffer (with-out phenylmethylsulfonyl fluoride), divided into small aliquots,and frozen at -70°C. Each aliquot was thawed only once forfusion experiments.

Fusion of Reconstituted Vesicles with RBL Cells. To defineoptimal fusion conditions, 2 X 106 parental or variant RBL cellswere incubated with vesicles containing 10 ,g of total proteinin 200 ,ul of Tyrode buffer for 30 min at 4°C with occasionalshaking, during which time the vesicles attached to the cells(step 1). The fusion process was then induced by incubating theagglutinated cells at 37°C for 20 min (step 2). The cells werethen washed and suspended in RPMI 1640 medium containing10% heat-inactivated fetal calf serum and incubated for an ad-ditional 4 hr at 37°C for recovery (step 3). During this period,essentially all living cells became attached to the substrate. Thosecells that were detached were removed by washings and onlythe adherent ones were used in the following experiments.

Degranulation Measurements. The degranulation processwas monitored either by the fluorometric histamine determi-nation method of Shore et al. (18) or by a slight modification ofthe procedure for measuring [3H]serotonin release describedby Taurog et al. (19). In the latter method, 107 cells were in-cubated for 24 hr at 37°C in 10 ml of RPMI 1640 medium con-taining 1.2-2.5 X 10-6 mol of [3H]serotonin (10-20 Ci/mmol;1 Ci = 3.7 x 10'° Bq; Amersham). Cells were washed in me-dium to remove excess [3H]serotonin and were processed forfusion. After the 4-hr recovery period, cells were suspended bycareful pipetting and plated into 96-well flat-bottom microtiterplates (Nunclon), 105 cells in 100 Al of medium per well. Thecells were passively sensitized by incubating them with ascitic

fluid containing 2,4-dinitrophenyl (DNP)-specific monoclonalIgE antibodies (20), 150 ul of a 1:50 dilution per well for 1 hrat 40C. The supernatants were removed and the cells washedthree times with medium to remove unbound IgE. The cellswere challenged to release [3H]serotonin by adding 10 ;Zg ofthe antigen [a conjugate of eight molecules of DNP per onemolecule of bovine serum albumin, (DNP)8-BS albumin] to the150 1.I of medium per well, and plates were incubated for 30min at 37C. One-hundred-microliter samples were removedfrom each well into a toluene/Triton scintillation liquid for de-termination of the released [3H]serotonin. The total radiola-beled serotonin incorporated into cells was estimated by dis-solving the cells in 100 A.l of 1 M NaOH and counting theradioactivity in the whole sample. Release was expressed aspercentage of total [3H]serotonin incorporated.

Ca2" Influx Measurements. The Ca2" uptake studies utilizethe procedure of Crews et al. (21) with a slight modification.Samples of 106 passively sensitized cells loaded with [3H]sero-tonin were plated in 3.0 ml of medium on 35-mm (diameter)Petri dishes in 10 mM Hepes-buffered saline (pH 7.4) con-taining '45CaCl2 (at 10 puCi/ml; Amersham) in the presence of1 mM CaCl2 (final specific activity, 10 Ci/mol) and were stim-ulated for degranulation with 10 1Lg of (DNP)8-BS albumin perml for 30 min at 37°C. The medium was removed and assayed,either for histamine by the fluorometric method or for [3H]se-rotonin as described above. Ca2+ influx was stopped by wash-ing the attached cells three times with ice-cold saline containing10 mM Hepes, pH 7.4/10 mM CaCl2. One milliliter of hy-potonic 5mM CaCl2 solution was then added and the cells werelysed by repeated freezing and thawing. The thawed lysate wascollected and centrifuged to remove cellular debris. The ra-dioactivity of each aliquot was measured by liquid scintillation.

Analytical Procedures. Protein concentrations were deter-mined by the Peterson method (22) with bovine serum albuminas a standard. Slab gels of NaDodSO4/polyacrylamide lineargradients (7-15%) were made according to Laemmli (23). Sam-ples were reduced with 0.25 M mercaptoethanol before loadingon the gels. Isolated CBP was radioiodinated either by the chlo-ramine-T method (24) or with the Bolton-Hunter reagent (25).

RESULTSThe purified CBP was implanted into RBL-2H3 variant cellsdefective in their capacity for cromolyn binding, Ca2' uptake,and degranulation by using Sendai-virus envelope vesicles asthe fusogenic agent. The Ca2+ uptake and degranulation werethen measured in the reconstituted variant cells upon immu-nological stimulation.

Preparation and Characterization of VCBP Vesicles. To ob-tain efficient implantation, highly fusogenic vesicles were re-quired. To achieve this end we performed double-titration ex-periments, varying the range of both the CBP/vesicles ratio(5-75 ug of CBP per 5 mg of soluble viral envelopes) and thevesicle/cell ratio (1-30 ,ug of vesicle protein per 2 X 106 cells).The minimal amount of CBP required to induce a measurabledegranulation of the deficient variants was found to be 20 ,ug/5 mg of viral envelope protein. At the other extreme, no fur-ther enhancement in degranulation was noted when CBP con-centrations were increased beyond 50 ,ug/5 mg of viral en-velope protein. Therefore, the conditions chosen for reconsti-tution were 50 pug of CBP per 5 mg of viral envelopes. Underthese conditions, 80-90% of the purified CBP were reprod-ucibly incorporated into the VCBP vesicles, as determined byincluding "I-labeled CBP ('"I-CBP) as a tracer. Electropho-retic analysis of isolated CBP and VCBP vesicles (Fig. 1, lanesc-e and c'-e') showed that CBP indeed became an integralcomponent of the VCBP vesicles. The presence of the two viral

Immunology: Mazurek et al.

6016 Immunology: Mazurek et al.

NP

FIG. 1. NaDodSO4/polyacrylamide gel electrophoresis of CBP-in-corporated vesicles and their immunoprecipitates with specific anti-viral antiserum. Samples were reduced with 0.25 M mercaptoethanol.The low amount of CBP in the VCBP vesicles was not detectable byCoomassie blue staining of the gels (<1 pg); therefore '"I-CBP wasused (lanes c and d and c' and d'). (A) Coomassie blue staining of: lanea, immunoprecipitate of a mixture ofV vesicles and 125I-CBP with an-tiviral antiserum; lane b, V vesicles; lane c, immunoprecipitate of 125I-labeled VCBP (125I-VCBP) vesicles with antiviral antiserum; lane d,reconstituted 1"I-VCBP vesicles [the upper band is of the viral enve-lopeNH glycoprotein (65,000 daltons) and the lower band is ofthe viralenvelope F glycoprotein (53,000 daltons)]; lane e, isolated CBP con-taining 12'I-CBP added as a tracer; lane f, antiviral antiserum; lane M.W.,molecular mass markers (in daltons): phosphorylase b, 94,000; bovineserum albumin, 67,000; ovalbumin, 43,000; carbonic anhydrase, 30,000;trypsin inhibitor, 20,100; a-lactalbumin, 14,400. Whenever immuno-precipitation was performed, protein bands originating from the anti-serum are also seen on the gels (lanes a and c). (B) Autoradiogram ofthe Coomassie blue-stained gel. Lanes a'-e' of the autoradiogram cor-respond to lanes a-e of the Coomassie blue-stained gel inA; lane g, iso-lated CBP that was iodinated by the Bolton-Hunter reagent. (In all othercases, CBP was radiolabeled by the chloramine-T method.) The 24,000-dalton protein (seen in lanes e, c', d', e', and g) appears consistently,though in different amounts, in the preparations of CBP prepared byeither affinity chromatography or by immunoprecipitation (13).

envelope glycoproteins (glycoprotein NH, 65,000 daltons, andprotein F, 53,000 daltons) can clearly be observed in the elec-trophoretic patterns of the VCBP vesicles (Fig. 1, lanes c andd).To further ascertain that the CBP was integrated into the

VCBP vesicles, anti-Sendai-virus antiserum was used to im-munoprecipitate both V vesicles and VCBP vesicles. More than75% of the labeled CBP incorporated into the VCBP vesiclescould be precipitated by this antiserum, whereas no radioac-tivity was precipitated when only 125I-CBP was used. More-over, when 125I-CBP was mixed (but not incorporated) with theviral (V) vesicles, <1% of the radioactivity was precipitated.Upon analysis by NaDodSO4/polyacrylamide gel electropho-resis, the immunoprecipitate of a mixture of 125I-CBP and Vvesicles with antiviral antiserum showed only bands corre-

sponding to the virus and to the antiviral antiserum (Fig. 1,lanes a and b and a' and b'). In contrast, in the autoradiogram,the VCBP precipitated with the same antibodies showed a pat-tern similar to that of the isolated CBP (Fig. 1, lanes c and e

and c' and e').Fusogenic Properties of VCBP Vesicles. Labeled VCBP ves-

icles were examined for their ability to fuse with CBP-deficientRBL-2H3 variant cells. When 125I-CBP alone or in a mixturewith V vesicles was tested (2 ,tg of labeled protein alone or mixedwith 10 ,g of V vesicles, yielding a final concentration of 0.01%Triton X-100 in the mixture) only a minor (<2%) amount of thelabeled CBP was retained with the cells. Each preparation ofVCBP vesicles was assayed for the precise optimal fusogenicdose (Table 1); with doses above 20 ,ug of envelope protein,vesicle-induced cell-cell fusion was observed after extensivelysis. Below 5 ,ug of envelope protein, no cell-cell fusion was

observed. Most efficient vesicle-cell fusion as well as an ac-

Table 1. Incorporation of CBP into RBL-2H3 variant cells asconstituent of the viral vesicles or in a mixture with them

Total 1251I-CBP incorporatedvesicle during the fusion Cell

Type of protein, process, viability,vesicles Ag Step it Step 2 Step 3§ %*

15I-VCBP 2.5 42 19 14 955.0 47 24 18 907.5 65 34 29 85

10 85 55 47 8515 73 23 27 8020 48 22 16 70

V + 125I-CBP 10 3 2 2 8510 2 1 1 90

25I-CBP 2 1 <1 <1 90

Three different preparations ofCBP and viral envelope proteins havebeen used in separate and independent experiments. Radioactivity in-corporated into the host cells is presented as % of the total 125I-VCBPinput.* Cell viability was determined at the end of the process (4 hr after fu-sion at 370C) by trypan blue exclusion. The recovery of viable cells is-65% of that prior to fusion (see text). The results given are the meanvalues of four independent experiments and differed by less than ±5%.

tAt 40C for 30 min.tAt 370C for 20 min.§ At 370C for 4 hr.

ceptable low level of cell-cell fusion and lysis were found in therange of 7.5-10 Ag of viral protein per 2 x 106 cells. In ad-dition, cell viability remained 85% under these conditions (Ta-ble 1). At this optimal vesicle concentration, up to 90% of theradioactivity incorporated into the VCBP vesicles was found tobe associated with the host cells after 30 min at 40C. Upon in-cubation of the cells for 20 min at 370C, conditions required forthe integration of vesicles into the host-cell membrane (26), theamount of label on the host cells was reduced to 30-45%. Nofurther change in the amount of radioactivity was observed duringovernight incubation.

Ca2' Influx and Degranulation in the Parental, Variant, andReconstituted Cells. The results shown in Table 2 demonstratethat upon IgE-mediated stimulation, untreated variant cells aswell as those fused with V vesicles exhibited a minimal increaseof Ca2' influx (<10% of the maximal signal) and as low as a 3%increase in histamine release. In contrast, VCBP-fused variantcells showed a marked increase in Ca2+ influx (up to 6,000 cpmper 106 cells relative to 650 cpm per 106 cells in the basal stateand to 6,200 cpm per 106 cells in the parental line). In addition,there was an increase in the histamine release from the fusedcells (up to 85% of the amount released by the parental line).That these represent CBP-mediated Ca2' influx and histaminerelease is supported by the fact that cromolyn and monoclonalanti-CBP antibodies shown to inhibit degranulation of the pa-rental cells also inhibited completely the Ca2' influx and his-tamine release induced in the VCBP-fused variants (Table 2).Furthermore, fusion-blocking antiviral antisera added to thevariant cells mixed with VCBP at 40C prevented the inductionof CBP-mediated Ca2P influx on subsequent incubation at 370C.In contrast, addition of antiviral antisera after fusion at 370Cfailed to forestall the CBP-mediated degranulation process. Thisdemonstrates that the active fusion of the vesicles is a condi-tional requirement for the induction of the CBP-mediated Ca2+influx. Titrations monitoring the Ca2+ influx and histamine re-lease as a function of the amount of CBP incorporated into theRBL variants (Fig. 2) show a sigmoidal dependence.

Proc. Natl. Acad. Sci. USA 80 (1983)

Proc. Natl. Acad. Sci. USA 80 (1983) 6017

Table 2. Ca2" uptake and degranulation of reconstituted variants upon immunological triggeringInhibition of degranulation by

Cells reconstituted with donor vesicles Monoclonal anti-CBPUnreconstituted cells V vesicles VCBP vesicles Cromolyn* antibodiestCa2" Ca2" Ca2" Ca2" Ca2+

uptake, uptake, uptake, uptake, uptake,cpm/30 Histamine cpm/30 Histamine cpm/30 Histamine cpm/30 Histamine cpm/30 Histaminemin per release, min per release, min per release, min per release, min per release,

Host cells 106 cells % 10i cells % 106 cells % 106 cells % 10' cells %ParentalRBI2H3 6,090 30 - - - 2,450 8 1,330 1

VariantsRBL-2H3-E7-14 1,370 3 1,490 2 6,270 26 2,150 6 1,350 2RBL-2H3-E7-23 675 0 690 0 5,900 24 2,100 7 770 0

Cells grown for 48 hr were sensitized with a monoclonal, DNP-specific IgE for 1 hr at 40C and the unbound IgE was removed by several washingswith RPMI medium. Ca2+ (1 mM Ca2+; 10 Ci/mol) was added 10 min prior to the challenge with (DNP)8 BS albumin for 30 min at 370C. The su-pernatant was sampled for degranulation and the cells were assayed for calcium uptake. The basal uptake (mean + SD) of Ca2+ was 650 ± 50 cpmper 106 cells and the spontaneous histamine releasewas3% oftheir content. Net histamine release data are presented. All data given are the averageof five independent experiments. In three experiments, degranulation was followed by histamine determination by using the fluorometric assayof Shore et al. (18) and, in two other experiments, it was followed by measuring [3Hlserotonin release. The standard deviations for Ca2+ uptake were+10%, for histamine release they were ±8%, and for [3H]serotonin release they were ±4%.* Inhibition of degranulation by cromolyn was achieved by incubating the cells in the presence of 100 /AM cromolyn 5 min prior to challenging themwith (DNP)s-BS albumin.

t One million cells were preincubated with 100 ,ul of supernatant of hybridoma culture for 1 hr at 40C; they were then washed and challenged with(DNP)8-BS albumin.

DISCUSSIONImplantation of CBP into the variants resulted in restoration ofclose to 85% of the Ca2' uptake and degranulation capacity ob-served in the parental cell line. It has been reported that Ca2'introduced into mast cells by Ca2'-loaded liposomes inducesdegranulation in the accepting cells (27). Induction of degran-ulation due to adventitious cell damage or vesicular Ca2' trans-port was excluded in our work by allowing the reconstitutedcells a 4-hr recovery period prior to the immunological trig-gering and by fusion with V vesicles, respectively. The specificinvolvement of CBP in restoration of degranulation in the re-constituted variants was examined by utilizing specific inhib-itors, such as cromolyn or specific monoclonal anti-CBP anti-bodies. The latter were shown to inhibit Ca2' uptake anddegranulation also in the parental RBL-2H3 cells.

Functional restoration is found to be dependent on the amount

0

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0)

c

E

(n

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5D06

-5 Efro

-4 I;n-'0

-3ECcx

,c'in

CBP incorporated x IO15g/cefll

FIG. 2. Reconstitution ofCa2" uptake (A) and degranulation (o) uponimplantation ofvarying amounts ofCBP into the RBL-2H3-E7-14 vari-ant cells. To change the amount of incorporated CBP, variant cells (2x 106) were fused with the same amount (10 ,ug) ofVCBP vesicles con-

taining different concentrations of CBP; thus, the efficiency of fusionshould be the same throughout the titration. Each point represents anexperiment done in duplicate with standard deviations of ±6% for his-tamine release and ± 10% for Ca2" uptake. The basal levels of hista-mine release and Ca2" uptake are 3% and 675 cpm, respectively. Thesewere substracted from each point presented.

of CBP implanted into the variants: only low response was notedin cells containing 2 X I05 CBP molecules per cell, whereas avirtually full response was found in cells containing 8 x 105 CBPmolecules per cell (Fig. 2). Both Ca2' uptake and degranulationseem to show a sigmoidal response to the amount of CBP in-corporated into the variant cells. Such a sigmoidal response curvewould support cooperative involvement of more than a singleCBP molecule in its function. More data are naturally required;yet, a preliminary analysis of those presented in Fig. 2 in termsof the Hill formalism provides an n = 2 value. This implies thattwo CBP molecules are required for the step leading to themonitored functions of the cells.

In conclusion, the data presented here demonstrate that bothCa2' uptake and degranulation of the CBP-defective variantsare solely due to implantation of CBP into the membranes ofthese variants. Moreover, it demonstrates that CBP plays a cru-cial role in the Ca2' gating process. Future work should at-tempt to resolve whether CBP is the Ca2' gate itself, a con-stitutional subunit of it, or a regulatory protein in Ca2+ influxmechanism.

The devoted help of Nechama Zakai and Yehudit Laster is gratefullyacknowledged. This work was partly supported by Grant 7349 to A. L.from the U. S.-Israel Binational Science Foundation.

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